Monitoring and Measuring Mammalian Autophagy
Macroautophagy (autophagy) is a conserved lysosomal-based intracellular degradation pathway. Here, we present different methods used for monitoring autophagy at cellular level. The methods involve Atg8/LC3 detection and quantification by Western blot, autophagic flux measurement through Western blot, direct fluorescence microscopy or indirect immunofluorescence, and finally traffic light assay using tf-LC3-II. Monitoring autophagic flux is experimentally challenging but obviously a prerequisite for the proper investigation of the process. These methods are suitable for screening purposes and can be used for measurements in cell lysates as well as in living cells. These assays have proven useful for the identification of genes and small molecules that regulate autophagy in mammalian cells.
KeywordsAutophagic flux LC3-II p62 TEM tf-LC3
Research support was partly provided by the Department of Biotechnology [Grant Number: BT/PR7791/BRB/10/1187/2013], Board of Research in Nuclear Sciences (BRNS) [Number: 37(1)/14/38/2016-BRNS/37276], Department of Atomic Energy (DAE), and Science and Engineering Research Board (SERB) [Number: EMR/2016/001246]. Research infrastructure was partly provided by Fund for Improvement of S&T Infrastructure in Universities and Higher Educational Institutions (FIST) [Number: SR/FST/LSI-025/2014], Department of Science and Technology, Government of India.
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